The Golgi complex is a central membrane organelle for intracellular trafficking, glycosylation and sorting of membrane and secretory proteins. The basic structure of the Golgi is a stack of flattened cisternae, but how this structure is assembled and inherited during cell division is poorly understood. In the last few years, we have developed a unique multidisciplinary approach employing biochemistry, cell biology, electron microscopy, and more recently proteomics and glycomics, combined with a novel in vitro reconstitution assay, to provide a mechanistic explanation for Golgi structure formation and function. We found that GRASP55 and GRASP65 play complementary and essential roles in Golgi structure formation by forming mitotically regulated trans- oligomers that hold the Golgi membranes into stacks. Using GRASP55/65 as tools to manipulate Golgi stack formation, we provided the first evidence that Golgi stacking impedes protein trafficking to ensure accurate post-translational modifications. We hypothesize that the GRASP proteins play essential roles in Golgi structure formation through oligomerization and interaction with other proteins, which subsequently regulates protein trafficking and glycosylation. This proposal is a logical continuation of our previous studies to assess how the Golgi structure is assembled and why it is important for protein trafficking, glycosylation and sorting. The specific aims are: 1) Identify and characterize novel GRASP65 interacting proteins that regulate Golgi structure formation. Our preliminary data showed that there are proteins in the interphase cell cytosol that enhance GRASP65 oligomerization. We have identified 20 proteins that interact with GRASP65 and we will characterize their roles in Golgi structure formation. This will also help us understand how GRASPs perform multiple functions as previously reported. 2) Determine the structure- function relationship of the Golgi in protein trafficking, glycosylation, and sorting. We will manipulate th Golgi structure by knocking out GRASP55/65, using the recently developed CRISPR/Cas9 technique, and by expressing GRASP55/65 mutants in cells to determine the consequences of Golgi destruction and restoration on protein trafficking, modifications and secretion. Significantly, alterations in Golgi structure and function have been associated with a variety of human diseases, including cancer, autoimmune disease, viral infections, and Huntington's and Alzheimer's diseases. Golgi defects may affect the trafficking, sorting and modification of a large number of proteins and cause global effects inside the cell and on the cell surface that compromise a variety of cellular functions. A better understanding of Golgi structure formation and the relationship to its vital cellular function is required before its role in human disease ca be understood. Our proposed study will determine the consequence of Golgi destruction on protein trafficking and processing, and thus provide fundamental information on the role of the Golgi under normal conditions and the relationship between Golgi defects and disease pathogenesis.